DESCRIPTION

IBA1, also known as AIF1, is a 17-kDa cytoplasmic calcium-binding protein belongs to the EF-hand calcium-binding protein family. IBA1 is widely used as the most common cytoplasmic protein for microglial immunostaining, with high sensitivity across a majority of microglial states in both white and gray matter. In the healthy CNS, IBA1-positive microglia perform immune surveillance, while following CNS insult-ranging from neurodegenerative protein deposits to chronic neuroinflammation-IBA1 expression is upregulated, marking activated microglia that enwrap amyloid plaques in Alzheimer's disease and contribute to the clearance of apoptotic cells and debris. Thus, IBA1 is not only a useful biomarker of microglial activation but also an active participant in regulating the innate immune functions of the brain, making it a potential target for therapeutic strategies aimed at modulating microglia-mediated neuroinflammation.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	8	48.58	4.18	8.60
2	8	12.75	0.36	2.82
3	8	7.55	0.33	4.37

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	48.84	4.30	8.80
2	16	12.87	0.41	3.19
3	16	7.64	0.35	4.58

RECOVERY

The recovery of human IBA1 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:8	98	88-105
	1:16	105	91-124

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	98
	Range (%)	94-102
1:8	Average% of Expected	84
	Range (%)	83-84
1:16	Average% of Expected	72
	Range (%)	71-74

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human IBA1 One-Step ELISA Kit produced at Sunlong Medical™.